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					RESEARCH REPORT

Fresh and cultured buccal cells as a source of mRNA and protein for molecular analysis
Agnes Michalczyk1, George Varigos2, Lance Smith3, and M. Leigh Ackland1
BioTechniques 37:262-269 (August 2004)

We developed a method for obtaining viable buccal cells from mouthwash samples for use as a source of mRNA and protein. Immunofluorescent analysis showed that most cells were derived from nonkeratinized parabasal epithelia, with a minor proportion of proliferative cells. Gene expression was detected in buccal cells using reverse transcription PCR, Western blot analysis, and immunofluorescence. Using a keratinocyte-specific medium, buccal cells could be cultured on Matrigel™-coated permeable filters for up to 2 weeks while maintaining the expression of some epithelial-specific markers, including cytokeratin 13, cytokeratin 10, transferrin receptor, and β-integrin. The basal marker cytokeratin 14 and Ki67, an indicator of cellular proliferation, were detected in a few cells. We show that buccal cells can be obtained from a noninvasive procedure for use as a source of material for biochemical analyses. A population of the buccal cells can be maintained in culture for up to 2 weeks using keratinocyte-specific medium in combination with extracellular matrix.

INTRODUCTION Human tissue is essential for investigations to determine the cellular and molecular basis of disease. However, access to patient cells can be a limiting factor due to the risk of trauma for patients, the need for involvement of qualified medical personnel in the sampling procedures, or the inaccessibility of tissue. Furthermore, it may not be possible to obtain a sufficient amount of tissue for analysis. The standard method for obtaining cells is through the collection of blood by venipuncture or tissue biopsy. Both these methods are invasive and patients may therefore refuse to participate in the study. Noninvasive methods of obtaining material could allow for distance collection where no medical persons are available and in screening initiatives as part of public health and preventative programs. Buccal cells are a source of material that can be obtained from noninvasive collection methods. They have been used to detect cancer-associated changes in the oral cavity using oral exfoliative cytology (1). Buccal cells have provided a source of DNA for analysis using PCR or other genotype tests (2–7). The DNA obtained from buccal cells is stable at room temperature
1Deakin

over a period of months (8). A range of methods has been used to obtain buccal cells for the analysis of DNA. These include using cotton swabs (9), cytobrushes (9–11), a “swish and spit” method (2,6,12), a modified Guthrie card (13), and a method of rubbing cheeks against teeth to exfoliate cells (14). Optimization of mouthwash solutions for buccal cell collection has improved yields of buccal cells (7,10,15). Cells from mouthwash may originate from a different part of the oral cavity and thus present various phenotypes; however, the identity of mouthwash cells has not been established. Despite the success of methods for isolating DNA from buccal cells, there are no reported methods for obtaining mRNA and protein from mouthwash cells. The analysis of mRNA and protein expression can provide important information of altered cellular function associated with inherited disorders. In this study, we describe a novel method for the isolation of mRNA from buccal cells obtained from mouthwashes. We show that mouthwash cells can be used for quantitative analysis using real-time PCR and Western blot analysis and for the detection of specific oral epithelial markers using immunofluorescence that have not been previ-

ously reported. We describe a novel culture method in which buccal cells can be maintained for up to 2 weeks, while retaining the expression of epithelial-specific markers. MATERIALS AND METHODS Collection of Buccal Cells Ethical approval for the collection of human cells, including buccal cells from mouthwash, was obtained from Deakin University (Melbourne, Australia) and the Royal Children’s Hospital (Parkville, Australia). Two mouthwashes were collected from each of eight participants. Prior to the collection of each sample, the participants’ mouths were rinsed with 20 mL Listerine® solution. Wash 1 consisted of cells obtained from each donor following vigorous brushing (the forcefulness of the brushing was the same as used for normal tooth brushing) of the inside of the oral cavity using a sterile toothbrush and was recovered from the donor mouths by rinsing with 15 mL deionized water. Additional cells were recovered by rinsing the brush with 10 mL deionized water, and both solutions were combined.

University, Melbourne, 2Royal Children’s Hospital, Parkville, and 3ThermoTrace, Noble Park, Australia
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After a 30-min break, each participant was asked for another sample (wash 2), which was collected following a similar protocol to wash 1. The buccal cells from both mouthwashes were pelleted by centrifugation at 3000× g for 5 min, rinsed three times with phosphate-buffered saline (PBS), and used immediately or frozen at -80°C. Cell Viability Tests Two methods were used to test for the viability of mouthwash cells. For the Trypan blue exclusion test, the freshly pelleted buccal cells were resuspended in 1 mL PBS. A 200-μL aliquot of Trypan blue solution (Sigma-Aldrich, Sydney, Australia) was mixed with 200 μL resuspended cells. The number of live (translucent) and dead (blue) cells was counted using a Brightline™ Hemocytometer (Hausser Scientific, Horsham, PA, USA) under phase-contrast microscopy (×100 magnification). For the Neutral red uptake test, freshly pelleted buccal cells were resuspended in 2 mL keratinocyte growth medium (KGM® 1; ThermoTrace, Melbourne, Australia) containing 0.05 mg/mL Neutral red stain and incubated for 3 h at 37°C. Live cells (which accumulated Neutral red dye) and dead cells (which did not absorb the dye) were counted using a hemocytometer. Images of cells stained by both methods were collected using an Olympus AX 70 microscope and RT Slider camera and Spot Advanced v. 3.0.4 software (Diagnostic Instruments, SciTech, Melbourne, Australia). RNA Preparation Frozen cell pellets from wash 2 were solubilized in 250 μL TE buffer [10 mM Tris-HCl, pH 8.0, 10 mM EDTA (Sigma-Aldrich)] containing 200 mM NaOH, and 1% sodium dodecyl sulfate (SDS) (High Pure Plasmid Isolation Kit; Roche Applied Science, Melbourne, Australia). Samples were then incubated for 5 min at room temperature. The solution was neutralized with 250 μL 3 M potassium acetate, pH 5.5, and incubated for 5 min at 4°C. The supernatant was collected by centrifugation at 18,000× g rpm for 10 min and used for total RNA extraction, following the manufacturer’s
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protocol (RNeasy® Mini Kit; Qiagen Pty. Ltd., Doncaster, Australia). DNA was removed from the samples using a DNA-free™ kit (Ambion, Houston, TX, USA). RNA concentrations and quality were estimated using a DU Series 500 Ultraviolet (UV)/Vis Spectrophotometer (Beckman Coulter, Fullerton, CA, USA) using a wavelength ratio of 260/280. Reverse Transcription PCR (RT-PCR) These procedures were carried out as described previously (16), using primers to GAPDH and the zinc transporter ZnT4 (SLC30A4) listed in Table 1. The primers for both ZnT4 and GAPDH spanned introns. Western Blot Analysis Buccal cell pellets from wash 2 were lysed by sonication at 15 pulses, 40% power output, 30% duty cycle, in

500 μL of 10 mM Tris-HCl buffer with 1% SDS (Sigma-Aldrich). Western blot analysis was carried out as previously described (16). Immunocytochemistry Freshly collected buccal cells were smeared onto a glass slide coated with 5% gelatine, left to dry for 5 min, and rinsed 3 times in PBS. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 5% TX-100 in PBS for 10 min, blocked with 3% bovine serum albumin (BSA) in PBS for 90 min, and immunofluorescence was carried out as previously described (17). Buccal Cell Cultures Freshly obtained mouthwash cells were rinsed once with PBS and incubated for 30 min in 500 U/mL penicillin and 500 μg/mL streptomycin solution with 12 μg/mL Fungizone (Sigma-

RESEARCH REPORT
Aldrich), and then washed twice with PBS. The cells were cultured for 4 weeks in 25 cm3 flasks, using 5 different growth media to test survival and growth: MCDB 153 (Sigma-Aldrich); KGM 1 (KGM supplemented with Fetuin); KGM 2 (KGM supplemented with chondroitin-4 sulphate); Xten™ GO; and Thermo minimal essential medium (TMEM) with 2% fetal bovine serum (FBS; ThermoTrace). Specifications for the noncommercial media KGM 1 and KGM 2 are provided as supplementary material at http://www. BioTechniques.com.August2004/ MichalczykSupplementary.html. All of the growth media contained 50 U/mL penicillin and 50 μg/mL streptomycin solution with 1.2 μg/mL Fungizone. Viability tests were performed on all of the cultures at day 1 and day 30. Freshly collected buccal cells were also seeded on Matrigel™ (Integrated Sciences, Sydney, Australia) spread on 24 mm diameter Transwell® filters (Corning Costar; EK Medical, Melbourne, Australia) and grown in KGM 1 or TMEM with 2% FBS media for 2 weeks. Filters with cells were then collected and processed for immunocytochemistry as described in the previous section.
Table 1. Primers Used for Reverse Transcription PCR Primer name ZnT4-C ZnT4-D ZnT4-E ZnT4-F β-actin-F β-actin-R Orientation F R F R F R Primer Sequence 5′-GGAAGCGCCTCAAATCTATGCT-3′ 5′-TACATTCAAAATGGCTTGGCACA-3′ 5′-GATAGCCTGGCAGTGAGAGCTG-3′ 5′-ATACGGACACAGCTGTCAGGGA-3′ 5′-ATCCCATCACCATCTTCCAG-3′ 5′-CCACCACCCTGTTGCTGTAG-3′ PCR Product Length (bp) 1004

610

750

Primers to ZnT4 were designed to GenBank® sequence accession no. AF025409. Primers to β-actin were designed to GenBank sequence accession no. E00829. F, forward; R, reverse.

Figure 1. Viable cell count of cells from fresh mouthwash samples. (A) Trypan blue exclusion test shows unstained live cells (L) and dark-blue stained dead cells (D). (B) Neutral red incorporation test shows dark-red live cells (L) and lightly stained or unstained dead cells (D). 264 BioTechniques

Figure 2. Reverse transcription PCR (RT-PCR) analysis of mouthwash samples and cultured cells. (A) Detection of GAPDH cDNA in extracts from the mouthwashes of two participants. Lane a, molecular weight marker (DNA Molecular Weight Marker XIV; Roche Applied Science); lane b, participant 1 mouthwash with GAPDH-F and GAPDH-R primers; lane c, participant 2 mouthwash with GAPDH-F and GAPDH-R primers; lane d, control with no cDNA; lane e, control with no reverse transcription sample. (B) Detection of hZnT4 expression in extracts from the mouthwashes of two participants. Lane a, molecular weight marker; lane b, participant 1 mouthwash (ZnT4-E and ZnT4-F primers); lane c, participant 2 mouthwash (ZnT4-E and ZnT4-F primers); lane d, participant 1 mouthwash (ZnT4-C and ZnT4-D primers); lane e, participant 2 mouthwash (ZnT4-C and ZnT4-D primers). (C) Detection of GAPDH cDNA in cultured buccal cells. Lane a, molecular weight marker; lane b, participant 1 mouthwash with GAPDH-F and GAPDH-R primers; lane c, participant 2 mouthwash with GAPDH-F and GAPDH-R primers. Vol. 37, No. 2 (2004)

RESEARCH REPORT
RESULTS AND DISCUSSION Viability of Buccal Cells To obtain the maximum numbers of viable buccal cells, we used a toothbrush to remove surface epithelial cells, which were then collected following a mouthwash. Mouthwashes have previously been shown to give more and better quality (higher molecular weight) DNA compared to that obtained when a dry cytobrush is used to remove epithelia (10,14). Before the cells were collected, participants rinsed their mouths with Listerine. Commercial antibacterial solutions such as Listerine contain a significant amount of alcohol (21.6% alcohol in Listerine), which can significantly reduce the bacterial content of the collected sample, reducing the risk of contamination in further analysis and allowing for longer sample storage (6,7). The viability of mouthwash cells collected from eight participants was assessed for two separate washes: wash 1, which included vigorous mouth brushing with a toothbrush and rinsing with sterile water to obtain cells and wash 2, where cells were collected similarly but after a 30-min break. For each participant, the Trypan blue exclusion test (Figure 1A) and the Neutral red incorporation test (Figure 1B) were used to determine the number of live and dead cells from each mouthwash. The mean total number of cells obtained for all participants was 16.1 ± 4.1 × 105 for wash 1 and 17.2 ± 2.3 × 105 for wash 2. There was no significant difference between wash 1 and wash 2 (P > 0.05; t-test). The average number of live cells estimated by both methods for wash 1 was 1.1 ± 0.3 × 105, a viability of 7.2 ± 1.6%, with values ranging from 5.2% to 9.8%. For wash 2, the average number of live cells was significantly higher and determined to be 2.7 ± 0.4 × 105, a viability of 17.6 ± 3.1%, with values ranging from 13.5% to 22.6%, (P < 0.05, t-test). This indicated a significant increase in the viability of cells collected after the second brushing relative to the first brushing. The viability

Figure 3. Western blot analysis of mouthwashes of two participants. (A) β-actin protein was detected using monoclonal anti-β-actin antibody. Lane a, 60 μg of participant 1 mouthwash extract showing a strong 42-kDa band; lane b, 60 μg of participant 2 mouthwash extract also showing a 42-kDa band. (B) hZnT4 protein was detected using CSK antibody, raised to synthetic peptide. Lane a, 60 μg of participant 1 mouthwash extract showing a 47-kDa band of the expected size; lane b, 60 μg of participant 2 mouthwash extract showing a 47-kDa band of the expected size. 266 BioTechniques

Figure 4. Detection of the oral epithelium-specific markers in freshly collected mouthwash cells. (A) Cytokeratin 13 was found in the majority of cells (ck13); nuclei were stained with bisbenzamide (N). (B) Few cells expressed cytokeratin 10 (ck10), showing nuclear label with bisbenzamide only. (C) Cytokeratin 16 (ck16) was present in only a minority of cells; nuclei stained with bisbenzamide. (D) Transferrin receptor protein (Tf) was detected in a high proportion of the cells; nuclei stained with bisbenzamide. (E) Parabasal/basal marker integrin β1 (i) was found in many buccal cells; nuclei stained with bisbenzamide. Vol. 37, No. 2 (2004)

of buccal cells from mouthwashes has previously been estimated to be 44% to 75% (17,18) using Trypan blue staining, which is higher than our results. As most of the cells from the mouthwash are fully differentiated and have a very thick cell membrane (17), this may prevent Trypan blue penetration even if the cells are dead and could account for the high percentage of live cells seen in other studies. To confirm our Trypan blue results, we used Neutral red, which has to be actively transported into the cell. This gave similar results to the Trypan blue staining. Total RNA Isolation and RT-PCR The mRNA from wash 2 was used as a source of RNA for RT-PCR, as this wash contained a higher proportion of live cells compared to wash 1. Following PCR, GAPDH fragments of the expected size (750 bp) were detected (Figure 2A, lanes b and c). No bands were seen from the “no DNA” control (Figure 2A, lane d) or the “no reverse transcription” control (Figure 2A, lane e). The amplification from a low abundance mRNA (zinc transporter hZnT4) was also determined using two sets of primers (Table 1). Bands of the expected size of 610 bp (Figure 2B, lanes d and e) and 1004 bp (Figure 2B, lanes b and c) were detected on agarose gel. Western Blot Analysis A sufficient amount of buccal cell extract could be obtained from one collection to carry out protein analysis by Western blot analysis. For two participants, the amount of total protein obtained from the cells of wash 2 was estimated to be 3.6 and 3.1 mg/sample. Prior treatment of cells with SDS and sonication was required to solubilize proteins. The expression levels of β-actin protein in mouthwash from the two participants were measured by Western blot analysis using commercial β-actin antibody. A band of the expected size of 42 kDa was obtained in extracts from mouthwash cells (Figure 3A). hZnT4 protein was detected at low levels in buccal cells, as found previously (16). A band of the expected size of 47 kDa was found in both participants’ mouthwashes (Figure 3B).
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Immunocytochemistry To establish the identity of the cells from mouthwash, immunocytochemistry was used to detect the presence of specific oral epithelial markers. Specific proteins expressed in buccal cells could be immunolocalized if cells were permeabilized with a relatively high concentration (5%) of TX-100 prior to indirect immunostaining. Cytokeratin 13 protein was detected in a

large proportion of freshly prepared mouthwash cells (Figure 4A). This indicated that they were most likely derived from the buccal mucosa because cytokeratin 13 is a major protein expressed suprabasally in nonkeratinized buccal epithelium (19). The keratinizing epithelium marker cytokeratin 10 was visualized in few of the cells (Figure 4B). Cytokeratin 10 is a keratin found in the suprabasal layers of keratinizing epithelia and is

Figure 5. Immunocytochemical analysis of buccal cells maintained on Matrigel for 18 days. (A) Cytokeratin 13 (ck13) was detected in the majority of cells. (B) Cytokeratin 10 (ck10), (C) cytokeratin 16 (ck16), (D) transferrin receptor (Tf), and (E) integrin β1 (i) were detected in some cells. (F) Ki67 (k) and (G) cytokeratin 14 (ck14) were found in few cells. BioTechniques 267

RESEARCH REPORT
Table 2. Viability of Buccal Cells Cultured in Different Media After 4 Weeks Media Typea MCDB 153 KGM 1 KGM 2 Xten GO TMEM with 2% FBS Total Cell Count (cells/mL) 6.4 × 104 6.1 × 104 5.6 × 104 3.5 × 104 4.6 × 104 Live Cell Count (cells/mL) 1.8 × 104 4.8 × 104 2.5 × 104 1.4 × 104 3.0 × 104 Live Cells as Percent of Initial 9.5 25.3 13.2 7.4 15.8

TMEM, Thermo minimal essential medium; FBS, fetal bovine serum. aStarting total cell count for all the media types was 8 × 105 with 1.9 × 105 live cells.

also detected to a lesser extent suprabasally as stacks in nonkeratinizing buccal epithelia (19). Similarly, cytokeratin 16, a marker for high cell turnover characteristic for both keratinizing and nonkeratinizing types of epithelium, was only detected in a minority of cells (Figure 4C). The transferrin receptor, a protein characteristic of proliferating cells from the parabasal and basal layers of the epidermis

(20), was present in a high proportion of cells (Figure 4D). Another parabasal/basal layer marker, integrin β1, was also expressed by many mouthwash cells (Figure 4E), indicating a parabasal origin. Culturing of Mouthwash Cells To determine the optimal conditions for their survival, mouthwash cells

were cultured under different conditions. Prior to culturing buccal cells, several steps were taken to reduce the risk of infection. Participants were asked to rinse their mouths with Listerine solution. The freshly collected buccal cells from wash 2 were rinsed with high concentrations of antibiotics plus Fungizone. Cells were cultured in 25 cm3 flasks at a concentration of 4 × 105 with five different growth media (MCDB 153, KGM 1, KGM 2, Xten GO, and TMEM with 2% FBS) to test the suitability of each media type. After four weeks in culture, no attached cells were found in any of the media, but a small number of viable floating cells were detected. The highest survival rate of cultured cells was recorded for KGM 1 and TMEM media, where live cell counts of 4.8 × 104 and 3.04 × 104 were recorded (Table 2). To increase their survival, mouthwash cells were placed onto Transwell filters coated with Matrigel and incubat-

ed in either KGM 1 or TMEM with 2% FBS media. After 18 days, Trypan blue and Neutral red tests showed that 91% (KGM 1) and 87% (TMEM with 2% FBS) of the total cells present were viable in both of these media. ThermoTrace media were formulated to improve cell survival in a range of mammalian cell types. KGM 1 was specifically aimed at improving the proliferation of keratinocytes, while TMEM is an improved Eagle’s media formulation enriched to allow improved cell proliferation at reduced serum supplementation [2% FBSsupplemented TMEM was equivalent or better than 10% FBS-supplemented standard Dulbecco’s modified Eagle’s medium (DMEM)]. With reduced serum and modifications to calcium levels, it was expected that isolated primary cells would be less prone to cell differentiation (21,22). RT-PCR analysis detected the expression of GAPDH transcripts (Figure 2C), which confirmed cell viability. Adherent cells were analyzed by immunofluorescence for the presence of epidermal specific markers. Markers expressed in cultured adherent mouthwash samples were similar to those expressed in freshly isolated cells. Cytokeratin 13 was found in a majority of the cultured cells (Figure 5A). Cytokeratin 10 (Figure 5B), cytokeratin 16 (Figure 5C), transferrin receptor (Figure 5D), and integrin β1 (Figure 5E) were expressed in only a few cells. Ki67, a protein produced by proliferating cells such as those present in the parabasal and basal regions of epithelium, was detected in very few cells (Figure 5F), as was cytokeratin 14, another marker of the basal layer (Figure 5G). Overall, the marker profile of the cultured cells was similar to that of the fresh cells. Interestingly, Ki67 was found in a few cells, suggesting that the culture conditions were to some extent conducive to cell proliferation. It is also possible that the culture conditions promoted the survival of basal cells, as cytokeratin 14 was found in a few of the cultured cells but not detected in fresh cells.
ACKNOWLEDGMENTS

COMPETING INTERESTS STATEMENT

L.S. is employed by ThermoTrace, Biosciences Division, Thermo Electron Corporation, the manufacturer of some materials used in this study. A.M., G.V., and M.L.A. declare no competing interests.
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Received 12 December 2003; accepted 26 April 2004.
Address correspondence to: M. Leigh Ackland Deakin University Biological and Chemical Sciences 221 Burwood Highway Burwood, Melbourne VIC 3125 e-mail: leigha@deakin.edu.au BioTechniques 269

This study was supported by funding from the Australian Research Council to M.L.A.
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